| Abstract: | Nitric oxide (NO) is a regulatory molecule involved in many biological processes. NO is produced by nitric oxide synthase by conversion of l‐ arginine to l‐ citrulline. l‐ Arginine methylated derivatives, asymmetric and symmetric dimethylarginines (asymmetric dimethylarginine, ADMA, and symmetric dimethylarginine, SDMA), regulate l‐ arginine availability and the activity of nitric oxide synthase. As such, they have been frequently investigated as potential biomarkers in pathologies associated with dysfunctions in NO synthesis. Here, we present a new multistep analytical methodology based on liquid chromatography combined with mass spectrometry for the accurate identification of l‐ arginine, l‐ citrulline, ADMA and SDMA. Compounds are measured as stable 2,3,4,5,6‐pentafluorobenzoyl chloride derivatives, which allows for simultaneous analysis of all compounds through chromatographic separation of ADMA and SDMA using a reverse‐phase column. Serum aliquots (100 μL) were spiked with isotope‐labeled internal standards and sodium carbonate buffer. The derivatization process was carried out at 25°C for 10 minu using pentafluorobenzoyl chloride as derivatization reagent. Calibration demonstrated good linearity (R 2 = 0.9966–0.9986) for all derivatized compounds. Good accuracy (94.67–99.91%) and precision (1.92–11.8%) were observed for the quality control samples. The applicability of the method was evaluated in a cohort of angiological patients and healthy volunteers. The method discerned significantly lower l‐ arginine and l‐ citrulline in angiologic patients. This robust and fast LC‐ESI‐MS method may be a useful tool in quantitative analysis of l‐ arginine, ADMA, SDMA and l‐ citrulline. |
